Dieldrin modifies the hydrolysis of PIP2 and decreases the fertilization rate in Bufo arenarum oocytes

Camp. Biochem. Physiol. Vol. lIZC, No. 1, pp. 61-67, 1995 Copyright 0 1995 Elsevier Science Inc. Printed in Great Britain. All rights reserved 0742~8413/95 %9.50+ .OO

Pergamon

Dieldrin modifies the hydrolysis of PIP, and decreases the fertilization rate in Bujb arenarum oocytes Teresa M. Fonovich de Schroeder’ and Ana M. Pechen de D’Angelo Libiquima, Facultad de Ingenieria, (8300) Neuquen, Argentina

Universidad

National de1 Comahue, Buenos Aires 1400,

Carbachol treatment in Bclfo arenarum oocytes decreases the radioactivity in [32P]PIP, in the following 20 min after stimulation and increases the [3H]glycerol labeling of 1,ZDAG at 1 min of stimulation. On the contrary, in Dieldrin treated oocytes carbachol stimulation produces an increase in [32P]PIP, labeling without changes in [3H]1,2-DAG radioactivity. The sustained hydrolysis of PIP, observed in Control oocytes is necessary to generate the intracellular second messengers which initiate the fertilization pathway. The lack of response to muscarinic stimulation in Dieldrin treated oocytes, may be associated with an early activation of PIP,-PLC by the insecticide, producing a depletion of the PIP, pool previous to the stimulation with carhachol. These changes take place simultaneously with a decrease in the ability of Bufo arena-urn oocytes to be fertilized in vitro, suggesting a correlation between impairment in the PIP, cascade and a decrease in the fertilization rate. Key words: Dieldrin; Phosphatidylinositol4J Fertilization; Oocyte; Phosphatidylinositol turnover; Carbachol. Comp. Biochem.

bis phosphate; 1,Zdiacylglycerol; Phospholipase; 4 phosphate; Kinase; Phosphatidylinositols

Physiol. 112C, 61-67, 1995.

Introduction Organochlorinated cyclodienic insecticides had been extensively used in the past for different purposes. Residues from these xenobiotics can be found nowadays in water and soil, as the consequence of their high persistence, which is due to their extremely low degradability (Matsumura, 1975). Besides the toxic effects that

cyclodienic insecticides exert on the nervous system of different species, some other nocive effects have been reported on mammalian reproduction (Welch et al., 1971; Kupfer, 1975). Iwamatsu (1989) and Miyazaki (1988b; 1989) described the involvement of intracellular second messengers in the fertilization process. The PI cycle is involved in the response of many different cells to external stimulation through the generation of intracellular second messengers: IP, and 1,ZDAG (Abdel-Lattif, 1986; Rana and Hokin, 1990). The first one is known to interact with intracellular receptors, promoting Ca*+ release from internal stores to the cytoplasm (Clapper and Lee, 1985; Berridge, 1987), while 1,ZDAG is a well known PKC activator (Nishizuka, 1989). The hydrolysis of PIP, had been reported in oocytes from different species in response to external stimulation (McIntosh and Catt,

to: Dr. Ana M. Pechen de D’Angelo, Libiquima, Facultad de Ingenieria, Universidad Nacional de1 Comahue, Buenos Aires 1400, (8300) Neuquen, Argentina. Tel: 54 99 428 820. ‘Present addiess: Prosivad, Fat. Farmacia y Bioquimica, Junin 953, 7” piso, (1414) Buenos Aires, Argentina. Abbreviations: PI: phosphatidylinositol; PIP: phosphatidylinositol4 phosphate; PIPr: phosphatidylinositol4J bis phosphate; 1,2-DAG: 1,Zdiacylglycerol; IPr: inosito1 1,4,5 trisphosphate; PLC: phospholipase C; PKC: nrotein kinase C. Received 31 August 1994; revised 24 April 1995; 27 April 1995. Correspondence

61

62

T. M. Fonovich de Schroeder and A. M. PechCn de D’Angelo

1987; Bernard et al., 1988; Rana and Hokin, 1990). More recently the expression of mRNAs coding for different oocyte Go subunits: Gao, Gai-1, Gai-3 and Gas, has been reported (Oiiate et al., 1992). Gao is the most abundant specie and may probably regulate the PLC activity in response to acetylcholine in these cells. Microinjection of IP, and GTP-y-S produced cortical granule exocytosis in Medaka eggs (Iwamatsu, 1989) as well as a rise in intracellular Ca2+ concentration in Medaka (Iwamatsu, 1989) and Golden hamster (Miyazaki, 1989) eggs. Bement and Capco (1989) demonstrated that phorbol 12-myristate 13-acetate (PMA) and Ca*+ ionophore A23187 synergistically trigger cortical granule exocytosis in Xenopus laevis oocytes, suggesting that PKC activation is an integral component of the fertilization pathway. We have already reported some alterations produced by prolonged Dieldrin treatment of Bufo arenarum oocytes, on the PI cycle. The results of that study led us to suggest that the pesticide may probably act on these cells through the inhibition of some enzyme activities, related to the synthesis of phospholipids and phosphatidylinositols (Fonovich de Schrodeder and Pechen de D’ Angelo, 1991). The present work was performed to study Dieldrin effects on the muscarinic mediated hydrolysis of phosphatidylinositols and the generation of 1,2-DAG, in oocytes that have been briefly exposed to the insecticide.

Materials Adult Bufo arenarum males and females were collected from the province of Buenos Aires. Dieldrin (1,2,3,4,10,10-hexachloro-6, 7 - epoxi - 1,4,4a,5,6,7,8,8a- octa- hydroxy - exo 1,4-endo-5,8-dimethane-naphthalene) was a gift from Shell Compatiia Argentina de Petroleo and it was purified according to the method reported by Maule et al. (1987). [32P]P0,HNa2 (130 Ci/g PO,HNa,) was provided by CNEA (Comision National de Energia Atomica). 2[3H]glycerol (5 Ci/mmol) and [y-32PlATP (10 Ci/mmol) were obtained from New England Nuclear. Phosphatidylinositol as well as acylglycerol standards were purchased from Sigma. Other chemicals were of analytical grade and solvents were of chromatographic grade.

Methods 1) Oocytes labeling Adult Bufo arenarum females were simultaneously injected with an homologous hypo-

physis homogenate and 60 t.&i of 2-[3H]glycerol in 1 ml of ethanol: H,O (1: 19) solution; 16 hr after the injection (when the female began to extrude the oocytes) the toad was phyted and oocytes were removed by surgery from the ovisacs. The oocytes were dejellied by 0.01 M thioglycolic acid treatment. Approximately 10,000 oocytes were incubated in two separate groups: Controls (in Ringer’s solution) or Dieldrin treated cells (in 4 pg/ml of Dieldrin in Ringer’s solution) during 2 hr. The ratio media/biota was 0.1 ml/oocyte. When the incubation was finished the cells were gently washed and incubated an hour more in 90 ml of 20 mM buffer TRIS containing 5.4 mM KCl, 140 mM NaCl, 1.8 mM CaCl,, 0.8 mM MgSO, and 35 PCi [32P]P0,HNa2, pH = 7.4. 2) Oocytes stimulation Prelabeled oocytes were gently washed and exposed to 1 mM carbachol in 20 mM buffer TRIS containing 5.4 mM KCl, 140 mM NaCl, 1.8 mM CaCl,, 0.8 mM MgS04, pH = 7.4, during different periods of time: 0, 1 and 20 min. 3) Extraction and chromatography

of lipids

Oocytes were homogenized with 40% trichloroacetic acid (TCA), centrifuged and the pellets were washed with 5% TCA. Lipids twice with chloroform : were extracted methanol (2: 1) in order to obtain phospholipids and neutral lipids. The resulting pellet once with chloroform : was reextracted methanol:12 N HCl (1OO:lOO:l) and then with chloroform : methanol : 12 N HCl (200 : 100: 1) with 20 min of sonication, in order to obtain phosphatidylinositols. separation. Acidic Phosphatidylinositois extracts were washed with 0.1 N HCl, chloroform : methanol : 0.1 N HCl and chloroform : methanol:O.Ol N HCl (3 :48:47), they were neutralized with 14 N NH,OH and concentrated under nitrogen stream to 1 ml per sample. Aliquots from each sample were taken to measure phospholipidic phosphorus (Pi) content (Rouser et al., 1970). After solvent removal phosphatidylinositols were resuspended in chloroform : methanol (2 : 1) and they were separated by thin layer chromatography on silica gel 60 H plates previously coated with 1% K+-oxalate, using chloroform: methanol:4 N NH,OH (90:70:20) as the mobile phase (Rodriguez de Turco and Spitzer, 1991). PI, PIP and PIP2 bands were identified by co-chromatography with standards on each plate. Samples were visualized by iodine vapors and they were scrapped into vials containing 10 ml of a scintillation cock-

Dieldrin effect on PIPz hydrolysis and fertilization

tail. Radioactivity was assayed on a Beckman 3801 liquid scintillation counter. I ,2-DAG separation. Chloroform : methanol (2: 1) extracts were washed twice with 0.88% KC1 and twice with chloroform : methanol : 0.88% KC1 (3 : 48 : 47). Samples were dried by a stream of nitrogen and resuspended in chloroform : methanol (2 : 1). Phospholipidic phosphorus content was measured as described previously and 1,2-DAG was separated by thin layer chromatography on silica gel 60 G plates, with hexane : diethyl ether : acetic acid (60: 40: 2.5) as the mobile phase (Pechen and Bazan, 1977). The samples were visualized by I, vapors and 1,2-DAG was identified by comparison with standards. The bands were scraped off and radioactivity was assayed as described above. 4) Fertilization Recently obtained [ 3H]glycerol-labeled oocytes, with or without their jelly coat, were in vitro inseminated in order to record the ability of the nonstored cells to be fertilized. Groups of at least 100 oocytes each were in vitro inseminated in Petri dishes by duplicate. Both groups were equally successful and fertilization rate was above 60%. Control and Dieldrin-treated oocytes were also inseminated by the same procedure after 2 hr of exposure to the insecticide, or 3 hr (2 hr with the insecticide plus 1 hr of exposure to [32P]P0,HNa,). Inseminations of jellied-oocytes were carried out in Ringer solution, while inseminations of dejellied ones were performed by addition of sperm suspension (lo6 cells/ml) in Ringer-phosphate (0.05 M) buffer, pH = 7.4 (Fonovich de Schroeder and Pechen de D’ Angelo, 1993). 5) Phosphatidylinositol

kinase assay

Membrane preparation. Recently obtained Bufo arenarum oocytes were dejellied and homogenized in HEPES buffer (20 mM) pH = 7.4 containing 0.25 M sucrose, 2 mM ethylene glycol-bis(P-amino-ethyl ether) (EGTA), 2 mM MgCl, and the protease inhibitors phenylmethylsulfonyl fluoride (PMSF) (100 PM), dithiotreitol (1 mM) and mercaptoethanol (10 mM). The homogenate was centrifuged during 5 min at 250 x g and the resulting supernatant was again centrifuged, during 40 min at 11,000 x g. The pellet was resuspended in HEPES buffer (20 mM) pH = 7.4 and stored in liquid nitrogen. Protein concentration was determined by the method of Lowry et al. (1951) modified by Peterson (1979). Enzyme activity. The activity of the PI kinase was measured by quantitating the trans-

63

fer of phosphate from [y_32P]ATP to PI. The assay tubes contained: 20 mM HEPES buffer pH = 7.4, 2 mM MgCl, and 0.2 mM ATP, 20-30 pg of protein, Nonidet P40 (0.25%, w/ v) and 0.5 mM PI, in a final volume of 100 ~1. Nonidet P40 and PI were added 5 min prior to the addition of ATP. After 5 min of preincubation at 23°C the reaction was started by addition of [y-32P]ATP (0.13 $i/nmol) and MgCl,. Samples were incubated during 5 min at 23°C and reactions were stopped by the addition of 0.1 ml of 20% TCA containing 1 mM ATP. Inositol lipid standards (40 pg each) and bovine serum albumin (BSA) were added to each tube. Samples were chilled on ice for 20 min, centrifuged and the resulting supernatant was discarded. Pellets were washed with 5% TCA and extracted with chloroform : methanol : 12 N HCl(lO0 : 100 : 1) as described. [32P]PIP was separated from the acidic extract and its radioactivity was measured as described above. Dieldrin effect on the PI kinase activity was evaluated by addition of the purified insecticide in an acetone solution, to the reaction tubes. The solvent was evaporated at 23°C before the addition of the membrane. After 20 min of preincubation, the enzyme activity was assayed as described previously. ATP concentration was varied in order to study possible effects of the pesticide at different substrate concentrations, maintaining the MgCl,/ATP concentrations ratio in lO/ 1. The activity of the PI kinase was also assayed in membranes obtained from oocytes previously exposed to Dieldrin for 2 hr.

Results A decrease in [32P]PIP2 labeling was observed when nonstimulated Dieldrin-treated oocytes prelabeled with [3H]glycerol, were exposed to [32P]P0,HNa2 in buffer solution for 60 min. This result is not associated either with an impairment in PIP, synthesis or with its degradation by phosphatases, as PI and PIP were labeled to the same extent in nonstimulated Control and Dieldrin treated oocytes (Fig. 1). Figure 1 also shows [32P]phosphatidylinositols labeling during carbachol stimulation for different periods of time. PI and PIP showed no significant changes in the 32P radioactivity at the periods studied, both in Control and in Dieldrin treated cells. PIP, radioactivity decreased with stimulation in Control oocytes and a recovery phase was not observed during the following 20 min. This response is similar to the one we found working with recently obtained oocytes (Fonovich de Schroeder, 1993). On the contrary, Dieldrin treated oo-

T. M. Fonovich de Schroeder and A. M. Pechtn de D’Angelo

64

1 32P radloactivi ty (cpmhmol

H]V-DAG

Pi)

(cpm/lOOrcmolPI)

::::r

a 0

22

1

2000

Timetminutes)

PIP

m

Control

m

Dleldrl”

1500 r---

Fig. 2. [3H]glycerol 1,ZDAG labeling in Control and Dieldrin treated oocytes on basal conditions (time = 0) and during carbachol stimulation (times = 1 and 22 Fin). Results represent the mean of triplicate samples (X 2 SD) and are expressed as [‘HI 1,2-DAG radioactivity &pm/ 100 pmol Pi). Student t-test: a) p < 0.05 between 0 time and 1 min of stimulation in Control oocytes. 2000,

t

PI 1500

-

1000

Time ~Control

(minutes) m

Dlrldr,n

Fig. 1. Phosphatidylinositols labeling with [32P]P04HNa2 in [3H]glycerol prelabeled Control and Dieldrin treated Eufo arenarum oocytes, on basal conditions (time = 0) and during carbachol stimulation (times = 1 and 22 Fin). Results represent the mean of triplicate samples (X + SD) and are expressed as 32Pradioactivity (cpmiwol Pi). ANOVA: a) p < 0.05 between Control and Dieldrin treated cells PIP2 radioactivity at 22 min of stimulation; b) p < 0.05 for Dieldrin treated oocytes’ PIP2, between different stimulation periods of time.

cytes evidenced an increase in [32P]PIP, labeling, as a consequence of muscarinic stimulation. [3H]1 ,ZDAG radioactivity increased transiently at 1 min of carbachol stimulation in Control oocytes, while Dieldrin treated cells did not show any variation on 1,ZDAG labeling due to exposure to the same agonist (Fig. 2). These results are consistent with a phospholipase C-mediated PIP, degradation in Control oocytes and the lack of enzyme activ-

ity in cells previously exposed to the pesticide (Figs. 1 and 2). Table 1 illustrates the percentages of successful fertilization in Control and Dieldrin treated oocytes, prelabeled with [3H]glycerol or [ 3H]glycerol plus [32P]P0,HNa2. Dieldrin produced a marked decrease in the ability of 3H-prelabeled oocytes to be fertilized, after a period of 2 hr of exposure. The effect was magnified when an additional incubation of 60 min was included, in order to label the cells with [32P]P04HNa, before insemination. The assay of PI kinase from oocyte membranes is shown in Fig. 3. The activity was linear at all the protein concentrations studied. Figure 4 shows the results obtained when the activity of the PI kinase was studied in vitro, with or without the addition of different Dieldrin concentrations. The pesticide did not Table 1. Fertilization rates of Control and Dieldrin treated oocytes prelabeled with [3H]glycerol and incubated during 2 hr in Ringer’s solution or Ringer’s solution plus 4 pglml Dieldrin, as well as Control and Dieldrin treated oocytes handled as mentioned above and incubated an hour more with [32P]P0,HNa2-Results represent the mean (3 + SD) of duplicate samples containing about 100 cells each and are expressed as percentage of Control fertilized oocytes Treatment [3H]glycerol + 2 hr incubation [3H]glycerol + 2 hr incubation + 1 hr “P labeling

Control (%)

Dieldrin (%)

100.04 2 5.24

70.30 -c 8.08

100.04 2 20.72

42.65 -t 10.19

ANOVA: p < 0.025 between treatments.

65

Dieldrin effect on PIP2 hydrolysis and fertilization

ATP

‘: 0.008 ;; k LJI 0006.

E \ 5E OOOL : -c ;

0 000

c

0002

q

0.096

mM

I 1

q-+-

1

COfIbd

E L ,” 0000 0.

-‘9

-‘a

0

-7 log

16

-‘5

-IL

-3

(Ml

Dietdrln

ATP=

0138

mM

Fig. 3. PI kinase activity in Bufo arenarum oocyte membranes as a function of protein concentration. Results represent the mean of duplicate samples and are expressed as [‘*PIPIP formed (nmol) during 5 min of incubation.

affect the enzyme activity at any of the concentrations tested. Incubation of intact oocytes for 2 hr with 4 pg/ml Dieldrin before membrane preparation and enzyme assay, was also ineffective in producing any change on the PI kinase activity (Table 2).

Discussion David et al. (1988) reported that activation of sea urchin eggs after insemination, produced a rapid depolarization followed by a second phase of inward current, concomitantly with intracellular calcium transients that take place at fertilization. An outward current develops 2 min after fertilization. Those results cannot exclude the possibility that a separate anion channel may also be activated during fertilization. Thus, the electrical response of the oocyte to sperm attachment and incorporation, described in sea urchin, resembles the one observed by Kusano et al. (1977) upon stimulation of Xenopus laevis oocytes with acetylcholine. Oron et al. (1985) found the link between phosphatidylinositols metabolism and neurotransmitter-induced physiological response, when they demonstrated that intracellularly injected IP, mimicked the muscarinic depolarizing chloride current, in Xenopus laevis oocytes. From those former experiments of muscarinic stimulation of oocytes to date, the presence of native M, and M, muscarinic receptors has been reported in Xenopus oocytes (McIntosh and Catt, 1987; Matus-Leibovitch et al., 1992) and the involvement of the phosphatidylinositols signal transduction pathway in the fertilization process, has also been es-

L

-9

-7

-6

log

_‘6 Dleldrln

-5

-I4

-‘3

It-41

Fig. 4. Dieldrin effect on the PI kinase activity for different ATP-MgC& concentrations. Results represent the mean of duplicate samples and are expressed as [32P]PIP formed (nmollmg proteins) as a function of log Dieldrin concentration (M).

tablished (Miyazaki, 1988a; Miyazaki, 1989; Crossley et al., 1988; Iwamatsu, 1989; Bement and Capco, 1989; Heinecke and Shapiro, 1992). The aim of the present study was to test the hypothesis that cyclodienic pesticides could affect fertilization of the toad Bufo arenarum, by interfering with the PI cycle, when previously exposed oocytes were in vitro inseminated. The low level of [32P]PIP2 labeling that we found in Dieldrin treated oocytes at zero time of stimulation (Fig. l), without [32P]PIP or [32P]PI accumulation, constitutes a first eviTable 2. PI kinase activity of Byfo arenarum oocyte membranes, obtained from cells pretreated with 4 @g/ml Dieldrin in Ringer’s solution during -2 hr and Control ones-Results represent the mean (X r SD) of quadruplicate samples and are expressed as [3zP]PIP formed during 5 min of incubation (nmol/mg proteins) Membrane Source Control oocytes Dieldrin-treated oocvtes

PI Kinase Activity (nmol [32P]PIP/mg prot.) 0.067 + 0.006 0.069 + 0.002

66

T. M. Fonovich de Schroeder and A. M. Pechen de D’Angelo

dence for an in vivo phospholipase activation, probably mediated by the intercalation of the insecticide in the oocyte membrane. The activation of such a phospholipase, may be similar to the one we observed when we studied in vitro the activity of the phosphatidylcholine phospholipase C from Clostridium perfringens, using Bufo arenarum oocyte membranes previously treated with Dieldrin as the substrate (Fonovich de Schroeder, 1993). The sustained hydrolysis of [32P]PIP2 that we found in Control oocytes, agree with the need of a sustained production of IP,. This second messenger controls Ca” release from internal stores, which is also modulated by the cytoplasmic concentration and diffusion of the cation (DeLisle, 1991; DeLisle and Welsh, 1992; Peres et al., 1991; Lechleiter and Clapham, 1992). The increase that we observed in [ 32P]PIP2 labeling during muscarinic stimulation of Dieldrin treated oocytes, must be the physiological recovery of the PIP, basal level in those cells, after Dieldrin-mediated degradation of PIP,. We cannot assure that the phospholipase which is activated by the pesticide is a phospholipase C because [3H] 1,2-DAG labeling was not increased before muscarinic stimulation in Dieldrin treated oocytes. Yet, this hypothesis cannot be ruled out because subsequent DAG lipase activation may be responsible for the disappearing (during the 2 hr of Dieldrin exposure) of the 1,2-DAG formed, as occurs in other tissues (Rindlisbather et al., 1987). On the other hand, we did not find any effect of Dieldrin on the PI kinase activity. This result agrees with the ones reported by Pulido et al. (1992). The lack in the accumulation of [32P]PI that we observed in the briefly treated cells, confirms the activation of a phospholipase instead of the inhibition of the synthetic pathway that we observed as the consequence of prolonged pesticide treatment (Fonovich de Schroeder and Pechen de D’Angelo, 1991), or the activation of phosphatases, as the ones responsible for the results discussed above. Direct activation of PI specific phospholipase C in the presence of organophosphorus insecticides (Davies and Holub, 1983) or maitotoxin (Bernard et al., 1988), as well as phtisphatidylcholine phospholipase C by cisDiammine dichloroplatinum (II) (CDDP) treatment (Nishio et al., 1992), has been reported in rat brain microsomes, amphibian oocytes and PC-9 cells respectively. We can conclude that Dieldrin affects the reproduction of the toad, through a decrease in the ability of oocytes to be fertilized, as can be seen in Table 1. This effect may be a

consequence of phospholipase-mediated depletion of the PIP2 pool, caused by exposure of the cells to the pesticide. Adequate PIP, level maintenance is absolutely necessary for the response of the PI cycle to external stimulation. Thus, phospholipase activation is an early event of the toxic effects of Dieldrin on Bufo urenurum oocytes and plays an important role in Dieldrin-induced cytotoxicity. Acknowledgements-We

thank Dr. Elena B. Rodriguez de Turco for expert advice on the determination of the PI kinase activity. T. F. de S. is the holder of a fellowship from CONICET.

References Abdel-Latif A. A. (1986) Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers. Pharmacol. Rev. 38, 227-278. Bement W. M. and Capco D. G. (1989) Activators of Protein kinase C trigger cortical granule exocytosis, cortical contraction, and cleavage furrow formation in Xenopus laevis oocytes and eggs. J. Cell Biol. 108,885-892. Bernard V., Laurent A., Derancourt J., Clement-Durand M.. Picard A.. LePeuch C.. Berta P. and DorCe M. (1988) Maitotoxin triggers the cortical reaction and phosphatidylinositol-4,5_bisphosphate breakdown in amphibian oocytes. Eur. J. Biochem. 174, 655-662. Berridge M. J. (1987) Inositol lipids and cell proliferation. Biochim. Biophys. Acta 907, 33-45.

Clapper D. L. and Lee H. C. (1985) Inositol trisphosphate induces calcium release from nonmitochondrial stores in sea urchin egg homogenates. J. Biol. Chem. 260, 13947-13954. Crossley I., Swann K., Chambers E. and Whitaker M. (1988) Activation of sea urchin eggs by inositol phosphates is independent of external calcium. Biochem. J. 252, 257-262.

David C., Halliwell J. and Whitaker M. (1988) Some properties of the membrane currents underlying the fertilization potential in sea urchin eggs. J. Physiol. 402, 139-154. Davies D. B. and Holub B. J. (1983) Comparative effects of organophosphorus insecticides on the activities of acetylcholinesterase, diacylglycerol kinase and phosphatidylinositol phosphodiesterase in rat brain microsomes. Pest. Biochem. Physiol. 20, 92-99. DeLisle S. (1991) The four dimensions of calcium signaling in Xenopus oocytes. Cell Calcium 12, 217-227. DeLisle S. and Welsh M. J. (1992) Inositol trisphosphate is required for the propagation of calcium waves in Xenoous oocvtes. J. Biol. Chem. 267. 7963-7966. Fonovich de Schroeder (1993) Efecto de1 Dieldrin sobre la transducci6n de setiales en ovocitos de sapo bufo arenarum, Hensel. Doctoral thesis, Universidad Nacional de Buenos Aires, Argentina. Fonovich de Schroeder T. M. and Pechtn de D’Angelo A. M. (1991) Dieldrin effects on phospholipid and phosphoinositide metabolism in Bufo arenarum oocytes. camp.

Biochem.

Physiol. 98C, i87-292.

Fonovich de Schroeder T. M.. Gauna L. and Pechtn de D’Angelo A. M. (1993) Is buffering capacity the principal role of the jelly coat in Bufo arenarum fertilization? Camp. Biochem.

Physiol. 105A, 533-537.

Heinecke J. W. and Shapiro B. M. (1992) The respiratory burst oxidase of fertilization. A physiological target for regulation by protein kinase C. J. Biol. Chem. 267, 7959-7962.

67

Dieldrin effect on PIP? hydrolysis and fertilization Iwamatsu T. (1989) Exocytosis of cortical alveoli and its initiation time in medaka eggs induced by micro injection of various agents. Develop. Growth & Differ. 31, 39-44. Kupfer D. (1975) Effects of pesticides and related compounds on steroid metabolism and function. CRC Critical Rev. Toxicol. 4, 83-124.

Kusano K., Miledi R. and Stinnakre J. (1977) Acetylcholine receptors in the oocyte membrane. Nature 270, 739-741. Lechleiter J. D. and Clapham D. E. (1992) Molecular mechanisms of intra cellular calcium excitability in Xenopus laevis oocytes. Cell 69, 283-294. Lipinsky D.. Oron Y. and Sarne Y. (1993) Morphine inhibits calcium influx and the response to acetylcholine in Xenapus oocytes. J. Neurochem. 60, 299-304. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. S. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Matsumura F. (1975) Toxicoloav of Insecticides. 2nd Ed. (Edited by Matsumura F.) I&rum Press. New York. Matus-Leibovitch N., Mengod G. and Oron Y. (1992) Kinetics of the functional loss of different muscarinic receptor isoforms in Xenopus oocytes. Biochem. J. 285, 753-758.

Maule A., Plyte S. and Quirk A. V. (1987) Dehalogenation of organochlorine insecticides by mixed anaerobic microbial populations. Pest. Biochem. Physiol. 27, 229-236.

McIntosh R. P. and Catt K. J. (1987) Coupling of inositol phospholipid hydrolysis to peptide hormone receptors expressed from adrenal and pituitary mRNA in Xenopus laevis oocytes. Proc. Natl. Acad. Sci. USA 84, 9045-9048.

Miyazaki S-I (1988a) Fertilization potential and calcium transients in mammalian eggs. Develop. Growth & Dif-

Nishizuka Y. (1989) The family of protein kinase C for signal transduction. J. Amer. Med. Assoc. 262, 1826-1833.

Odate A., Herrera L., Antonelli M., Bimbaumer L. and Olate J. (1992) Xenopus laevis oocyte Gn subunits mRNAs. Detection and quantitation during oogenesis and early embryogenesis by competitive reverse PCR. FEBS Letters 313, 213-219. Oron Y., Dascal N., Nadler E. and Lupu M. (1985) Inosi-

tol 1,4,5_trisphosphate Xenopus oocytes.

mimics muscarinic response in

Nature 313, 141-143.

Pechen A. M. and Bazan N. G. (1977) Lipid metabolism in early development using labeled precursors incorporated during oogenesis and in cell-free embryo homogenates. Lipids 12, 131-134. Peres A., Bertollini L. and Racca C. (1991) Characterization of Ca?’ transients induced by intra cellular photorelease of InsP, in mouse ovarian oocytes. Cell Calcium 12, 457-465.

Peterson G. L. (1979) Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal. Biochem. 100, 201-220. Pulido J. A., del Hoyo N. and Perez-Albarsanz M. A. (1992) The effects of different hexachlorocyclohexanes and cyclodienes on glucose uptake and inositol phospholipid synthesis in rat brain cortex. Life Sciences 50, 1585- 1596.

Rana R. S. and Hokin L. E. (1990) Role of phosphoinositides in transmembrane signaling. Physiol. Reviews 70, 115-164.

Rindlisbacher B., Reist M. and Zahler P. (1987) Diacylglycerol breakdown in plasma membranes of bovine chromaflin cells is a two-step mechanism mediated by diacylglycerol lipase and monoacylglycerol lipase. Biochim. Biophys. Acta 905, 349-357.

Rodriguez de Turco E. B. and Spitzer J. A. (1991) Hepatic fer. 30, 603-610. phosphatidylinositol kinase activity in continuously Miyazaki S-I (1988b) lnositol 1,4,5-trisphosphate-induced endotoxemic rats. Biochim. Biophys. Acfa 1093, 216222. calcium release and guanine nucleotide-binding proteinRouser G., Fleischer S. and Yamamoto A. (1970) Two mediated periodic calcium rises in golden hamster eggs. dimensional thin layer chromatographic separation of J. Cell Biol. 106, 345-353. polar lipids and determination of phospholipids by Miyazaki S-I (1989) Calcium wave in activating hamster phosphorus analysis of spots. Lipids 5,494-4%. eggs. Biol. Bull. 176(S), 21-24. Welch R. M., Levin W., Kuntzman R., Jacobson M. and Nishio K., Sugimoto Y., Fujiwara Y., Ohmori T., MoriConnev A. H. (1971) Effect of haloeenated hvdrocarkage T., Takeda Y., Ohata M. and Saijo N. (1992) Phosbon insecticides on the metabolism aid uterotropic acpholipase C-mediated hydrolysis of phosphatidylchotion of estrogens in rats and mice. Toxicol. Appl. Pharline is activated by cis-Diamminedichloroplatinum(I1). macol. 19, 234-246. J. Clin. Invesi. 89, 1622-1628.